Directed evolution of colE1 plasmid replication compatibility: a fast tractable tunable model for investigating biological orthogonality.

Plasmids of the ColE1 family are among the most frequently used plasmids in molecular biology. They were adopted early in the field for many biotechnology applications, and as model systems to study plasmid biology. The mechanism of replication of ColE1 plasmids is well understood, involving the interaction between a plasmid-encoded sense-antisense gene pair (RNAI and RNAII). Because of its mechanism of replication, bacterial cells cannot maintain two different plasmids with the same origin, with one being rapidly lost from the population — a process known as plasmid incompatibility. While mutations in the regulatory genes RNAI and RNAII have been reported to make colE1 plasmids more compatible, there has been no attempt to engineer compatible colE1 origins, which can be used for multi-plasmid applications and that can bypass design constrains created by the current limited plasmid origin repertoire available. Here, we show that by targeting sequence diversity to the loop regions of RNAI (and RNAII), it is possible to select new viable colE1 origins that are compatible with the wild-type one. We demonstrate origin compatibility is not simply determined by sequence divergence in the loops, and that pairwise compatibility is not an accurate guide for higher order interactions. We identify potential principles to engineer plasmid copy number independently from other regulatory strategies and we propose plasmid compatibility as a tractable model to study biological orthogonality. New characterised plasmid origins increase flexibility and accessible complexity of design for challenging synthetic biology applications where biological circuits can be dispersed between multiple independent genetic elements.

Viral Decoys: The Only Two Herpesviruses Infecting Invertebrates Evolved Different Transcriptional Strategies to Deflect Post-Transcriptional Editing

The highly versatile group of Herpesviruses cause disease in a wide range of hosts. In invertebrates, only two herpesviruses are known: the malacoherpesviruses HaHV-1 and OsHV-1 infecting gastropods and bivalves, respectively. To understand viral transcript architecture and diversity we first reconstructed full-length viral genomes of HaHV-1 infecting Haliotis diversicolor supertexta and OsHV-1 infecting Scapharca broughtonii by DNA-seq. We then used RNA-seq over the time-course of experimental infections to establish viral transcriptional dynamics, followed by PacBio long-read sequencing of full-length transcripts to untangle viral transcript architectures at two selected time points. Despite similarities in genome structure, in the number of genes and in the diverse transcriptomic architectures, we measured a ten-fold higher transcript variability in HaHV-1, with more extended antisense gene transcription. Transcriptional dynamics also appeared different, both in timing and expression trends. Both viruses were heavily affected by post-transcriptional modifications performed by ADAR1 affecting sense-antisense gene pairs forming dsRNAs. However, OsHV-1 concentrated these modifications in a few genomic hotspots, whereas HaHV-1 diluted ADAR1 impact by elongated and polycistronic transcripts distributed over its whole genome. These transcriptional strategies might thus provide alternative potential roles for sense-antisense transcription in viral transcriptomes to evade the host’s immune response in different virus–host combinations.

Computational analysis of sense-antisense chimeric transcripts reveals their potential regulatory features and the landscape of expression in human cells

Many human genes are transcribed from both strands and produce sense-antisense gene pairs. Sense-antisense (SAS) chimeric transcripts are produced upon the coalescing of exons/introns from both sense and antisense transcripts of the same gene. SAS chimera was first reported in prostate cancer cells. Subsequently, numerous SAS chimeras have been reported in the ChiTaRS-2.1 database. However, the landscape of their expression in human cells and functional aspects are still unknown. We found that longer palindromic sequences are a unique feature of SAS chimeras. Structural analysis indicates that a long hairpin-like structure formed by many consecutive Watson-Crick base pairs appears because of these long palindromic sequences, which possibly play a similar role as double-stranded RNA (dsRNA), interfering with gene expression. RNA–RNA interaction analysis suggested that SAS chimeras could significantly interact with their parental mRNAs, indicating their potential regulatory features. Here, 267 SAS chimeras were mapped in RNA-seq data from 16 healthy human tissues, revealing their expression in normal cells. Evolutionary analysis suggested the positive selection favoring sense-antisense fusions that significantly impacted the evolution of their function and structure. Overall, our study provides detailed insight into the expression landscape of SAS chimeras in human cells and identifies potential regulatory features.

The HIV-1 Antisense Gene ASP: The New Kid on the Block

Viruses have developed incredibly creative ways of making a virtue out of necessity, including taking full advantage of their small genomes. Indeed, viruses often encode multiple proteins within the same genomic region by using two or more reading frames in both orientations through a process called overprinting. Complex retroviruses provide compelling examples of that. The human immunodeficiency virus type 1 (HIV-1) genome expresses sixteen proteins from nine genes that are encoded in the three positive-sense reading frames. In addition, the genome of some HIV-1 strains contains a tenth gene in one of the negative-sense reading frames. The so-called Antisense Protein (ASP) gene overlaps the HIV-1 Rev Response Element (RRE) and the envelope glycoprotein gene, and encodes a highly hydrophobic protein of ~190 amino acids. Despite being identified over thirty years ago, relatively few studies have investigated the role that ASP may play in the virus lifecycle, and its expression in vivo is still questioned. Here we review the current knowledge about ASP, and we discuss some of the many unanswered questions.

Promoter switching in response to changing environment and elevated expression of protein-coding genes overlapping at their 5’ ends

Despite the number of studies focused on sense-antisense transcription, the key question of whether such organization evolved as a regulator of gene expression or if this is only a byproduct of other regulatory processes has not been elucidated to date. In this study, protein-coding sense-antisense gene pairs were analyzed with a particular focus on pairs overlapping at their 5’ ends. Analyses were performed in 73 human transcription start site libraries. The results of our studies showed that the overlap between genes is not a stable feature and depends on which TSSs are utilized in a given cell type. An analysis of gene expression did not confirm that overlap between genes causes downregulation of their expression. This observation contradicts earlier findings. In addition, we showed that the switch from one promoter to another, leading to genes overlap, may occur in response to changing environment of a cell or tissue. We also demonstrated that in transfected and cancerous cells genes overlap is observed more often in comparison with normal tissues. Moreover, utilization of overlapping promoters depends on particular state of a cell and, at least in some groups of genes, is not merely coincidental.

Human retroviral antisense mRNAs are retained in the nuclei of infected cells for viral persistence

Human retroviruses, including human T cell leukemia virus type 1 (HTLV-1) and HIV type 1 (HIV-1), encode an antisense gene in the negative strand of the provirus. Besides coding for proteins, the messenger RNAs (mRNAs) of retroviral antisense genes have also been found to regulate transcription directly. Thus, it has been proposed that retroviruses likely localize their antisense mRNAs to the nucleus in order to regulate nuclear events; however, this opposes the coding function of retroviral antisense mRNAs that requires a cytoplasmic localization for protein translation. Here, we provide direct evidence that retroviral antisense mRNAs are localized predominantly in the nuclei of infected cells. The retroviral 3′ LTR induces inefficient polyadenylation and nuclear retention of antisense mRNA. We further reveal that retroviral antisense RNAs retained in the nucleus associate with chromatin and have transcriptional regulatory function. While HTLV-1 antisense mRNA is recruited to the promoter of C-C chemokine receptor type 4 (CCR4) and enhances transcription from it to support the proliferation of HTLV-1–infected cells, HIV-1 antisense mRNA is recruited to the viral LTR and inhibits sense mRNA expression to maintain the latency of HIV-1 infection. In summary, retroviral antisense mRNAs are retained in nucleus, act like long noncoding RNAs instead of mRNAs, and contribute to viral persistence.

A study of the heterochronic sense/antisense RNA representation in florets of sexual and apomictic Paspalum notatum

Background Apomixis, an asexual mode of plant reproduction, is a genetically heritable trait evolutionarily related to sexuality, which enables the fixation of heterozygous genetic combinations through the development of maternal seeds. Recently, reference floral transcriptomes were generated from sexual and apomictic biotypes of Paspalum notatum, one of the most well-known plant models for the study of apomixis. However, the transcriptome dynamics, the occurrence of apomixis vs. sexual expression heterochronicity across consecutive developmental steps and the orientation of transcription (sense/antisense) remain unexplored. Results We produced 24 Illumina TruSeq®/ Hiseq 1500 sense/antisense floral transcriptome libraries covering four developmental stages (premeiosis, meiosis, postmeiosis, and anthesis) in biological triplicates, from an obligate apomictic and a full sexual genotype. De novo assemblies with Trinity yielded 103,699 and 100,114 transcripts for the apomictic and sexual samples respectively. A global comparative analysis involving reads from all developmental stages revealed 19,352 differentially expressed sense transcripts, of which 13,205 (68%) and 6147 (32%) were up- and down-regulated in apomictic samples with respect to the sexual ones. Interestingly, 100 differentially expressed antisense transcripts were detected, 55 (55%) of them up- and 45 (45%) down-regulated in apomictic libraries. A stage-by-stage comparative analysis showed a higher number of differentially expressed candidates due to heterochronicity discrimination: the highest number of differential sense transcripts was detected at premeiosis (23,651), followed by meiosis (22,830), postmeiosis (19,100), and anthesis (17,962), while the highest number of differential antisense transcripts were detected at anthesis (495), followed by postmeiosis (164), meiosis (120) and premeiosis (115). Members of the AP2, ARF, MYB and WRKY transcription factor families, as well as the auxin, jasmonate and cytokinin plant hormone families appeared broadly deregulated. Moreover, the chronological expression profile of several well-characterized apomixis controllers was examined in detail. Conclusions This work provides a quantitative sense/antisense gene expression catalogue covering several subsequent reproductive developmental stages from premeiosis to anthesis for apomictic and sexual P. notatum, with potential to reveal heterochronic expression between reproductive types and discover sense/antisense mediated regulation. We detected a contrasting transcriptional and hormonal control in apomixis and sexuality as well as specific sense/antisense modulation occurring at the onset of parthenogenesis.

Stress Induced Differential Expression of THAP9 & THAP9-AS1 in the S-Phase of Cell Cycle

Transposable elements (TE) function as one of the major effectors to respond to biological or environmental stress. The mobility of TEs, which is heavily controlled under normal conditions, may be activated by stress. LncRNAs are emerging as a crucial tool in the regulation of TEs. This study focuses on the gene expression of THAP9, a domesticated transposon and lncRNA THAP9-AS1 (THAP9-antisense1), which form a sense and antisense gene pair with a promoter overlap of approximately 350bp. Under basal conditions, THAP9 is preferentially transcribed while THAP9-AS1 is heavily down-regulated. In the S-phase of the cell cycle, THAP9 expression exhibits stress-specific effects ranging from moderate enhancement to no change. On the other hand, THAP9-AS1, which has previously been reported to be upregulated in several cancers, always demonstrates enhanced expression under stress. Moreover, THAP9-AS1 is transcriptionally favoured during stress since the stress-induced fold-increase of THAP-AS1 expression is always higher than THAP9. Interestingly, the expression of both THAP9 and THAP9-AS1 exhibit a striking periodicity throughout the S-phase, reminiscent of cell cycle regulated genes. Thus, this study sets the stage to further explore the relationship between THAP9 and THAP9-AS1 and investigate THAP9-AS1’s potential regulatory role during stress.

Transcription and splicing dynamics during early Drosophila development

ABSTRACTWidespread co-transcriptional splicing has been demonstrated from yeast to human. However, measuring the kinetics of splicing relative to transcription has been hampered by technical challenges. Here, we took advantage of native elongating transcript sequencing (NET-seq) to identify the position of RNA polymerase II (Pol II) when exons become ligated in the newly synthesized RNA. We analyzed Drosophila melanogaster embryos because the genes transcribed initially during development have few and short introns (like yeast genes), whereas genes transcribed later contain multiple long introns (more similar to human genes). We detected spliced NET-seq reads connected to Pol II molecules that were positioned just a few nucleotides downstream of the 3’ splice site. Although the majority of splice junctions were covered by spliced reads, many introns remained unspliced, resulting in a complex range of heterogeneity in splicing dynamics. Introns that show splicing completion before Pol II has reached the end of the downstream exon are necessarily intron-defined. As expected, we found a relationship between the proportion of spliced reads and intron size. However, intron definition was observed at all intron sizes. Both canonical and recursive splicing were associated with a higher Pol II density, suggesting a splicing-coupled mechanism that slows down transcription elongation. We further observed that transcription termination was very efficient for isolated genes but that the presence of an overlapping antisense gene was often associated with transcriptional read-through. Taken together, our data unravels novel dynamic features of Pol II transcription and splicing in the developing Drosophila embryo.

MORT, a locus for apoptosis in the human immunodeficiency virus-type 1 antisense gene: implications for AIDS, Cancer, and Covid-19

Apoptosis, or programmed cell death, is a fundamental requirement for life in multicellular organisms, including humans, and a mechanism to maintain homeostasis and prevent unwarranted cellular proliferations such as cancer. An antisense gene in HIV-1 (Hap) induces apoptosis in human cells. Apoptotic T cell death following HIV-1 infection leads to a compromised immune system and eventually AIDS (acquired immunodeficiency syndrome). A review of several studies that focused on long-term survivors of HIV-1 reveals that these survivors had deletion-mutations in Hap. A subset of these survivors changed course and experienced CD4+ T cell death and progression to AIDS. These individuals had virus that regained Hap gene sequence that had previously been deleted. Analysis of the changes in the genetic sequences with in vivo progression of the revertant HIV-1 virus allowed identification of a specific region in Hap we are calling MORT. MORT, in Hap RNA forms a primary microRNA-like structure. Potential human mRNAs targeted by MORT mi/siRNAs include gene/RNA sequences of X-linked inhibitor of apoptosis (XIAP), survivin, and apollon, along with many other human gene sites/RNAs. Thus MORT may be acting as an RNA antagonist to cellular IAPs thereby inducing apoptotic cell death. Surprisingly, additional potential MORT targets include viral sites in human SARS-CoV-2, including the protease, nsp5 RNA. Future uses for RNA therapy and a hypothesis for an HIV intrinsic mechanism utilizing MORT for viral anti-viral (or anti-microbial) and HIV anti-immune cell defense are proposed.